Metastasis is responsible for the majority of cancer-related deaths and prevention of metastasis remains a big challenge for cancer therapy. Cucurbitacin B (Cuc B) is a natural triterpenoid with potent anticancer activities while its effect on metastasis remains unclear. In the present study, the inhibitory effect and mechanisms of Cuc B on metastasis were investigated in MDA-MB-231 breast cancer cells. The cells were treated with or without Cuc B, and the cytotoxicity was determined by MTT assay. The effect of Cuc B on metastasis was evaluated with wound healing, transwell, and adhesion assays. Furthermore, the adhesion of cancer cells to endothelial cells was determined. The protein expression was determined by Western blotting. Cuc B (< 100 nmol·L) showed no obvious cytotoxicity to MDA-MB-231 cells, but significantly inhibited migration, invasion, and adhesion to Matrigel, fibronectin, type I collagen, and endothelial cells. Cuc B dramatically inhibited the phosphorylation of focal adhesion kinase (FAK) and paxillin in dose- and time-dependent manners. Furthermore, Cuc B induced intracellular reactive oxygen species (ROS) generation, which could be reduced by N-acetyl-l-cysteine (NAC). In addition, NAC pretreatment could reverse Cuc B-induced suppression of migration and adhesion, expression of FAK, but showed no effect on paxillin expression. In summary, Cuc B suppressed ROS-dependent metastasis through FAK pathway in breast cancer MDA-MB-231 cells, demonstrating novel mechanisms for the anticancer effects of Cuc B.
Acetylcysteine ; pharmacology ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; enzymology ; metabolism ; pathology ; physiopathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Collagen Type I ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Female ; Fibronectins ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Neoplasm Invasiveness ; pathology ; Neoplasm Metastasis ; pathology ; Paxillin ; metabolism ; Phosphorylation ; drug effects ; Reactive Oxygen Species ; metabolism ; Triterpenes ; antagonists & inhibitors ; chemistry ; pharmacology

BACKGROUNDAn acute respiratory distress syndrome (ARDS) is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase (JNK) on ARDS in a lipopolysaccharide (LPS)-induced ARDS rat model.

METHODSThirty-six rats were randomized into three groups: control, LPS, and LPS + JNK inhibitor. Rats were sacrificed 8 h after LPS treatment. The lung edema was observed by measuring the wet-to-dry weight (W/D) ratio of the lung. The severity of pulmonary inflammation was observed by measuring myeloperoxidase (MPO) activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid (BALF) were counted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was performed, and lung injury score was achieved in all three groups.

RESULTSMPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control (1.26 ± 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P < 0.05). Inhibiting JNK attenuated LPS-induced MPO activity upregulation (0.52 ± 0.12 U in LPS + JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P < 0.05). Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting JNK attenuated LPS-induced neutrophils increase in BALF (255.0 ± 164.4 in LPS vs. 53 (44.5-103) in control vs. 127.0 ± 44.3 in LPS + JNK inhibitor, P < 0.05). At the same time, the lung injury score showed a reduction in LPS + JNK inhibitor group comparing with that in LPS group (13.42 ± 4.82 vs. 7.00 ± 1.83, P = 0.001). However, the lung W/D ratio and the collagen in BALF did not show any differences between LPS and LPS + JNK inhibitor group.

CONCLUSIONSInhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis. JNK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

Animals ; Anthracenes ; therapeutic use ; Collagen ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Lipopolysaccharides ; toxicity ; Lung ; drug effects ; metabolism ; pathology ; Male ; Rats ; Respiratory Distress Syndrome, Adult ; chemically induced ; drug therapy ; Signal Transduction ; drug effects

BACKGROUND/AIMS: The development of therapeutic strategies for the treatment of cirrhosis has become an important focus for basic and clinical researchers. Adrenergic receptor antagonists have been evaluated as antifibrotic drugs in rodent models of carbon tetrachloride (CCl4)-induced cirrhosis. The aim of the present study was to evaluate the effects of carvedilol and doxazosin on fibrosis/cirrhosis in a hamster animal model. METHODS: Cirrhotic-induced hamsters were treated by daily administration of carvedilol and doxazosin for 6 weeks. Hepatic function and histological evaluation were conducted by measuring biochemical markers, including total bilirubin, aspartate aminotransferase, alanine aminotransferase and albumin, and liver tissue slices. Additionally, transforming growth factor beta (TGF-beta) immunohistochemistry was analyzed. RESULTS: Biochemical markers revealed that hepatic function was restored after treatment with doxazosin and carvedilol. Histological evaluation showed a decrease in collagen type I deposits and TGF-beta-secreting cells. CONCLUSIONS: Taken together, these results suggest that the decrease in collagen type I following treatment with doxazosin or carvedilol is achieved by decreasing the profibrotic activities of TGF-beta via the blockage of alpha1- and beta-adrenergic receptor. Consequently, a diminution of fibrotic tissue in the CCl4-induced model of cirrhosis is achieved.
Adrenergic alpha-1 Receptor Antagonists/*pharmacology ; Alanine Transaminase/blood ; Animals ; Aspartate Aminotransferases/blood ; Bilirubin/blood ; Carbazoles/*pharmacology ; Carbon Tetrachloride ; Collagen Type I/drug effects/metabolism ; Cricetinae ; Doxazosin/*pharmacology ; Liver/metabolism/pathology ; Liver Cirrhosis/blood/chemically induced/*drug therapy ; Liver Function Tests ; Propanolamines/*pharmacology ; Serum Albumin/analysis ; Transforming Growth Factor beta/blood/*drug effects

OBJECTIVETo investigate the inhibitory effects of OMT on TGF-β1-induced CFBs proliferation, and then explore the mechanism.

METHODThe experiment was randomly divided into 6 groups as following: control group (serum free DMEM), model group (20 μg x L(-1) TGF-β1), OMT low dose group (1.89 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), OMT medium dose group (3.78 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), OMT high dose group (7.56 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), SB203580 group (p38MAPK blocking agent, 1 x 10(-5) mol x L(-1) + 20 μg x L(-1) TGF-β1). Vimentin of CFBs was identified by immunocytochemical methods, α-SMA of myFBs as well. Inhibitory effects of OMT on CFBs proliferation was detected by the MTT assay. Picric acid Sirius red staining was analyzed collagen type I and collagen type III deposition. Western blot was determined the expression of p38MAPK, p-p38MAPK, collagen type I and collagen type III.

RESULTMTT results showed that OMT significantly inhibited CFBs proliferation induced by TGF-β1 (P < 0.01) α-SMA immunocytochemical experiments suggested that OMT could protect against the CFBs proliferation. OMT could significantly decrease the deposition of collagen type I and collagen type III by Western bloting and picric acid Sirius red staining. Western blot results showed that TGF-β1 enhanced p38MAPK phosphorylation, however OMT attenuated the phosphorylation of p38MAPK induced by TGF-β1 (P < 0.01).

CONCLUSIONOMT can inhibit the CFBs proliferation induced by TGF-β1, and its mechanism may be involved in inhibiting p38MAPK phosphorylation.

Alkaloids ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Collagen ; metabolism ; Down-Regulation ; Female ; Fibroblasts ; drug effects ; Heart ; drug effects ; In Vitro Techniques ; Male ; Phosphorylation ; Quinolizines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

BACKGROUND/AIMS: Our aim was to assess whether short-term treatment with soluble tumor necrosis factor (TNF) receptor affects circulating markers of bone metabolism in rheumatoid arthritis (RA) patients. METHODS: Thirty-three active RA patients, treated with oral disease-modifying antirheumatic drugs (DMARDs) and glucocorticoids for > 6 months, were administered etanercept for 12 weeks. Serum levels of bone metabolism markers were compared among patients treated with DMARDs at baseline and after etanercept treatment, normal controls and naive RA patients not previously treated with DMARDs (both age- and gender-matched). RESULTS: Bone-specific alkaline phosphatase (BSALP) and serum c-telopeptide (CTX)-1 levels were lower in RA patients treated with DMARDs than in DMARD-naive RA patients. After 12 weeks of etanercept treatment, serum CTX-1 and sclerostin levels increased. In patients whose DAS28 improved, the sclerostin level increased from 1.67 +/- 2.12 pg/mL at baseline to 2.51 +/- 3.03 pg/mL, which was statistically significant (p = 0.021). Increases in sclerostin levels after etanercept treatment were positively correlated with those of serum CTX-1 (r = 0.775), as were those of BSALP (r = 0.755). CONCLUSIONS: RA patients treated with DMARDs showed depressed bone metabolism compared to naive RA patients. Increases in serum CTX-1 and sclerostin levels after short-term etanercept treatment suggest reconstitution of bone metabolism homeostasis.
Adult ; Alkaline Phosphatase/blood ; Arthritis, Rheumatoid/blood/diagnosis/*drug therapy ; Biological Markers/blood ; Bone Morphogenetic Proteins/blood ; Bone Remodeling/*drug effects ; Collagen Type I/blood ; Female ; Genetic Markers ; Homeostasis ; Humans ; Immunoglobulin G/*administration & dosage ; Immunosuppressive Agents/*administration & dosage ; Inflammation Mediators/blood ; Male ; Middle Aged ; Peptides/blood ; Receptors, Tumor Necrosis Factor/*administration & dosage ; Time Factors ; Treatment Outcome ; Tumor Necrosis Factor-alpha/antagonists & inhibitors

PURPOSE: The present study was designed to determine whether rapamycin could inhibit transforming growth factor beta1 (TGF-beta1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway. MATERIALS AND METHODS: Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-beta1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay. RESULTS: Rapamycin significantly reduced TGF-beta1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-beta1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-beta1-mediated fibrogenic response in primary human lung fibroblasts. CONCLUSION: These results indicate that rapamycin effectively suppresses TGF-beta1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.
Cells, Cultured ; Collagen Type III/metabolism ; Fibroblasts/*drug effects/metabolism/physiology ; Fibronectins/metabolism ; Humans ; Lung/cytology/drug effects ; Pulmonary Fibrosis/drug therapy ; Signal Transduction/drug effects ; Sirolimus/*pharmacology ; TOR Serine-Threonine Kinases/metabolism/physiology ; Transforming Growth Factor beta1/*antagonists & inhibitors/physiology

OBJECTIVETo investigate the effects of spironolactone on the concentration of collagen type I, III in the myocardium of spontaneous hypertension rats (SHR).

METHODSTwenty 8-week male SHR were assigned randomly into spironolactone (SHR-SPIRO, n=10) and control groups (SHR-CON, n=10), sex-age matched Wistar Kyoto rats (WKY group, n=7) were also served as controls. The rats of SHR-SPIRO group were given 20 mg/(kg*d) of spironolactone, the rats of SHR-CON and WKY groups were given the same volume of distilled water. After 16 weeks, the concentration of collagen type I was analyzed with Western blot. The areas of collagen type I and III were observed under polarized light microscopy and the ratio of type I/III collagen was calculated through accumulation score.

RESULTSCompared with WKY group,the concentration of collagen type I in SHR-CON group was significantly higher (1.87 ±0.2 Compared with 1.21 ±0.7, P<0.05). After 16 weeks of treatment the concentration of collagen type I (1.42 ±0.05 Compared with 1.87 ±0.2, P<0.05) and I/III ratio in SHR-SPIRO group were significantly reduced (15.64 ±1.34 Compared with 20.8 ±3.04, P<0.05) compared with SHR-CON group; but there were no differences in accumulation area scores of collagen type III among three groups (368.3 ±30.2 Compared with 481.6 ±32.4 Compared with 406.2 ±45.3, P>0.05).

CONCLUSIONThe deposition of collagen type I in myocardium may be involved in myocardial fibrosis of SHR, and spironolactone can decrease the concentration of collagen type I, which may be one of the mechanisms for its therapeutic effects.

Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Male ; Mineralocorticoid Receptor Antagonists ; pharmacology ; Myocardium ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Spironolactone ; pharmacology

OBJECTIVE: To explore the inhibitory effect of calcitonin gene-related peptide (CGRP) on cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline and the underlying mechanism. METHODS: The primary cultured cardiac fibroblasts were incubated with isoprenaline (10(-5) mol/L) for 48 h after pretreatment with CGRP (10(-8) or 10(-7) mol/L) for 1 h. Cell activity was detected by MTT. The mRNA expression of collagen (types I and III) and connective tissue growth factor (CTGF) was determined by RT-PCR, and the levels of intracellular ROS were determined by DCFH-DA fluorescent probe. RESULTS: Isoprenaline significantly promoted fibroblast proliferation and up-regulated collagen (types I and III) and CTGF mRNA expression concomitantly with an increase in ROS production, which were attenuated by CGRP. The effect of CGRP on cardiac fibroblasts was inhibited by CGRP8-37, a selective antagonist of CGRP receptor. CONCLUSION CGRP is able to protect cardiac fibroblasts against isoprenaline-induced proliferation and collagen expression, which might be related to the down-regulation of CTGF expression through inhibition of ROS production.
Animals ; Animals, Newborn ; Calcitonin Gene-Related Peptide ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibroblasts ; cytology ; Isoproterenol ; antagonists & inhibitors ; pharmacology ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

This study aims to investigate the preventive role and potential mechanisms of blocking extracellular HMGB1 function on doxorubicin induced cardiac injury. Mice were treated with HMGB1 blocker glycyrrhizin 1 h before and one time every day (intraperitoneal, 10 mg per mouse) after doxorubicin injection, and sacrificed on the day 14 after doxorubicin challenge. Cardiac function was evaluated by echocardiography and hemodynamic measurement. Myocardial inflammation and collagen deposition were analyzed by immunohistochemistry and picrosirius red staining. The interaction of HMGB1 and TLR2 was assessed by co-immunoprecipitation and confocal microscopy. The protein contents of HMGB1, MyD88, p65NF-kappaB and phospho-p65NF-kappaB were measured by Immunoblot. Compared with mice treated with saline, doxorubicin treatment led to an upregulation in HMGB1 expression. Blocking HMGB1 activity with glycyrrhizin protected mice against cardiac dysfunction, inflammatory response, and cardiac fibrosis induced by doxorubicin challenge. Glycyrrhizin inhibited the interaction of HMGB1 and TLR2, and blocked the downstream signaling of TLR2. In conclusion, blocking HMGB1 protected against doxorubicin induced cardiac injury by inhibiting TLR2 signaling pathway.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Collagen ; metabolism ; Doxorubicin ; Drug Interactions ; Fibrosis ; Glycyrrhizic Acid ; pharmacology ; HMGB1 Protein ; antagonists & inhibitors ; metabolism ; Heart Diseases ; chemically induced ; metabolism ; pathology ; Immunoprecipitation ; Interleukin-17 ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Myocardium ; metabolism ; pathology ; Random Allocation ; Signal Transduction ; drug effects ; Toll-Like Receptor 2 ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Up-Regulation

This study is to determine the preventive effect and mechanism of targeting IL-17A on pulmonary inflammation and fibrosis after acute lung injury. Mice were treated with anti-IL-17A antibody on the day 7 and sacrificed on the day 14 after bleomycin lung injury. The pulmonary inflammatory status and the deposition of collagen were measured by HE and Sirius stains staining. The contents of hydroxyproline and collagen were measured by using commercial kits. The survival rate of mice was calculated by Kaplan-Meier methods. The inflammatory cytokines in bronchoalveolar lavage fluid were measured by ELISA and the expressions of inflammation-related molecules were detected by Western blotting assay. Targeting of IL-17A could prevent the development of lung inflammation, decrease collagen deposition and the contents of hydroxyproline, and protect against the development of pulmonary fibrosis, which together led to an increase in the animal survival. Moreover, blocking IL-17A decreased the expression ofpro-fibrotic cytokines such as IL-17A, TGF-beta1 and IL-13; increased the expression of anti-fibrotic or anti-inflammatory factors such as IFN-gamma, COX-2, 5-LOX, 15-LOX. Indeed, IL-17A antagonism suppressed the activation of pro-inflammatory p65NF-kappaB but enhanced the activation of pro-resolving p50NF-kappaB. In conclusion, that blockade of IL-17A prevents the development of pulmonary fibrosis from acute lung injury, is because blocking IL-17A may prevent acute inflammation converting to chronic inflammation.
Acute Lung Injury ; chemically induced ; complications ; Animals ; Bleomycin ; Collagen ; metabolism ; Hydroxyproline ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-17 ; antagonists & inhibitors ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B p50 Subunit ; metabolism ; Pneumonia ; etiology ; metabolism ; Pulmonary Fibrosis ; etiology ; metabolism ; prevention & control ; Random Allocation ; Transcription Factor RelA ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Up-Regulation